A mass spectrometer is an instrument that measures the charge-to-mass ratio of charged particles. Mass spectrometers are in widespread use in biochemistry laboratories to determine molecular weights of biomolecules, monitor bioreactions, detect post-translational modifications, perform protein and oligonucleotide sequencing, along with numerous other applications. One type of mass spectrometer, a matrix-assisted laser desorption ionization (MALDI) mass spectrometer, is particularly well suited for the mass spectrometric analysis and investigation of large molecules.
MALDI mass spectrometers utilize a method that allows for the vaporization and ionization of non-volatile biological samples from a solid-state phase directly into the gas phase. To do so, a sample (the “analyte”) is suspended or dissolved in a “matrix.” A matrix is a compound or ligand that may be co-crystallized with the analyte. It is reported that the presence of the matrix prevents the analyte from being degraded thereby allowing for the detection of intact molecules as large as 1 million Da.
A MALDI sample, typically in the form of a 2 mm or smaller diameter spot, is prepared by depositing a droplet of solution containing a solvent, the analyte, and the matrix on a flat surface and then permitting the droplet to dry. As this occurs, the matrix and the analyte co-crystallize on the surface. At times, the crystals that form are finely graduated and uniform in appearance, while at other times (depending on the matrix) the crystals may be irregular with visible crystalline “spears.”
During a MALDI experiment, a laser is focused on the MALDI sample spot. The laser functions as both the desorption and ionization source. In particular, the laser energy is absorbed by the matrix resulting in a microscopic explosion that creates a rapidly expanding matrix plume which carries both analyte and matrix into a vacuum where it is accelerated by an electric field and then transferred to a detector. The matrix also serves as a source of protons that facilitate the ionization of the analyte. The matrix molecules absorb most of the incident laser energy thereby reducing sample damage and ion fragmentation (i.e., soft ionization). Nitrogen lasers operating at prescribed wavelengths (e.g., a wavelength that is well absorbed by most UV matrices) are the most common illumination sources because they are inexpensive and offer a desired combination of power/wavelength/pulsewidth. However, other UV and even IR pulsed lasers have been used with properly selected matrices.
Once the analyte molecules are vaporized and ionized they are electrostatically transferred into a time-of-flight mass spectrometer (TOF-MS) where they are separated from the matrix ions and individually detected, based on their mass-to-charge (m/z) ratios, and thereafter analyzed. High transmission and sensitivity, along with theoretically unlimited mass range are among the inherent advantages of TOF instruments. Separation and detection of the ions at the end of the tube of the TOF instrument is based on their flight time, which is proportional to the square root of their mass-to-charge ratios.
It has been observed that the analyte signal intensity is highly dependent on the location in which the laser is focused on the MALDI sample spot. Certain regions of the MALDI sample spot produce strong analyte signals. Such regions are often referred to as “sweet spots.” In these sweet spot regions, the respective amounts of analyte and matrix are by chance proportioned to produce a strong, desirable signal. Moving the focus of the laser by a very small distance away from a sweet spot may significantly change the level of the observed analyte signal intensity. Note also that “sweet spots” are not necessarily long lived. Indeed, sample is released from the surface with every laser firing. As a result, “sweet spots” have a limited, unpredictable lifetime.
In typical experiments, the operator manually or remotely moves the sample around beneath the laser beam's focus while at the same time monitoring the signal intensity. When a strong signal is observed, the sample movement is stopped. The laser is then fired repeatedly (e.g., 5 Hz) with the results of each firing averaged to produce the final mass spectrum. The region around a “sweet spot” is often of great interest to the operator as acceptable signal intensity can often be found there. The sample throughput of such an operator-dependent technique is undesirably limited by sample handling requirements and the physical boundaries of operator speed. As such, the speed of sequentially interrogating MALDI sample spots has been limited by the natural limits of human reaction time. Indeed, it has been observed, for example, that an operator can manually trigger the laser, observe the results, determine whether the next spectrum should be acquired at the same target or a different target, move the sample spot (if necessary), and re-trigger the laser no faster than approximately once per second.